The overall purpose of this project is to investigate and develop a novel immune stimulant that can be used to elicit non-specific protection against pathogenic microorganisms that might be deployed in bioterrorist attacks. The product, comprised of cationic liposome-plasmid DNA complexes (CLDC), is an extremely potent stimulus for activation of innate immunity, particularly for release of interferons and other cytokines. The major advantages of CLDC relative to conventional immunostimulants are that they are more potent activators of interferon release, they can be administered by aerosol or mucosal delivery, they can be lyophilized and retain activity, and they are relatively simple and inexpensive to manufacture. The proposed studies are designed to test the hypothesis that CLDC can rapidly elicit sufficient protective innate immunity to be efficacious after parenteral or mucosal in an aerosol bacterial challenge rodent model. In preliminary studies, we have observed potent activation of innate immunity after administration of CLDC and efficacy after aerosol challenge with Mycobacterium tuberculosis. Complete protection has also been observed following intraperitoneal and intranasal administration of CLDC 48 hours prior to lethal intranasal challenge with Francisella tularensis LVS. We will, therefore, examine the effectiveness of CLDC-mediated immune activation by other routes of administration, particularly after mucosal delivery to the lungs and upper airways to evaluate protection against aerosol challenge with the more pathogenic F. tularensis Schu4. The specific goals of this project are (1) to determine how the route of administration and dosing regimen of CLDC affects the magnitude, quality, and duration of innate immune activation, and (2) determine the impact of CLDC-induced immunity on protection against F. tularensis challenge. We believe these studies will provide the basis for the CLDC's ability, in an easily administered format, to rapidly activate potent and consistent innate immune responses that will be essential in biodefense and civilian applications. [unreadable] [unreadable]